Protozoa are unicellular eukaryotic microorganisms that lack cell walls and are usually motile and colorless. They are distinguished from algae by their lack of chlorophyll, from fungi by their motility and absence of a cell wall, and from slime molds by their lack of fruiting body formation.
Protozoa are generally classified into four major groups based on their life cycles or mechanisms of motility: the flagellates, the cilliates, the amoeba, and the sporozoa (or Apicomplexa). The flagellates are protozoa that employ from one to eight or so flagella for movement. The ciliates employ cilia, which are shorter than flagella and present in large numbers. Protozoa that move by extending pseudopodia are called amoeba. The fourth major group, the sporozoa or Apicomplexa, are non-motile, intracellular parasites (except during their sexual stage) that penetrate host cells by a mechanism involving their characteristic apical complex. Some protozoa do not fit into any of these four groups, such as the non-motile, intracellular microsporidia, which penetrate host cells by an injection mechanism.
Clinically important representatives of the flagellate group include Giardia lamblia, Trichomonas vaginalis, Leishmania spp., and Trypanosoma spp. G. lamblia is a waterborne intestinal parasite that occurs worldwide, causing diarrhea, and other intestinal symptoms. The most commonly used drugs used to treat giardiasis are metronidazole and other members of the 5-nitroimidazoles. Unfortunately, Metronidazole is mutagenic in the Ames test (Vogd et al., Mutation Research, vol. 26, 483–490 (1974)) and has various toxic side effects. In addition, the development of resistance to these drugs in Giardia and other protozoan parasites such as Entamoeba histolytica and Trichomonas vaginalis also limits their effectiveness. Leishmaniasis, a life-threatening disease caused by Leishmania spp., is a major health problem worldwide with an estimated 10–15 million people infected and 400,000 new cases each year. There is currently no satisfactory treatment for leishmaniasis. The treatment of choice is pentavalent antimony in the form of sodium stibogluconate or meglumine antimonate. Both drugs are administered intravenously, have severe adverse side effects, require hospitalization during treatment, and are not always effective (M. Ouelette and B. Papadopoulou, Parasitology Today, vol. 9, pp. 150–153 (1993)). Trypanosoma spp. cause life-threatening diseases in humans, including African sleeping sickness and Chagas disease, as well as a number of important diseases in domestic animals. Leishmania and Trypanosoma are closely-related genera, representing the major pathogens in the kinetoplastid group of protozoa.
The ciliates are generally non pathogenic, except for Balantidium coli which is an intestinal parasite of domestic animals, in particular, swine. Occasionally, B. coli infects humans, producing a severe dysentery.
The amoeba group includes the intestinal parasite Entamoeba histolytica that causes amoebic dysentery and extraintestinal abscesses of organs such as the liver and lung. The most commonly used drug for treating E. histolytica infection is metronidazole. Other free-living amoeba, which occasionally cause infections in humans, include Acanthamoeba and Naegleria spp.; these infections are typically difficult to treat.
The sporozoa (also known as Apicomplexan parasites) are a large group of protozoa, all of which are obligate parasites and the pathogenesis of the diseases they cause are directly due to repeated cycles of host cell invasion, growth, and host cell lysis. Representative sporozoas are the malaria parasite Plasmodium spp.; the human water-born pathogen of worldwide medical importance, Cryptosporidium spp.; Toxoplasma gondii; and several parasites veterinary importance including Sarcocystis spp.; Theileria spp.; Babesia spp.; and Eimeria spp. (causing coccidiosis in fowl and domestic animals). Cryptosporidium parvum is a common cause of intestinal infection leading to self-limited diarrhea, but in the immunocompromized individual C. parvum infection is chronic and life-threatening. There is currently no effective treatment for cryptosporidiosis.
The numerous Apicomplexan parasitic protozoans seriously impact human health, livestock health and the economy. Toxoplasmosis is among the most common parasitic diseases of man. Serosurveys suggest prevalence rates as high as 70–90% in many areas of both the developing and developed world. Between 10–45% of Americans become infected at some point in their lives. An infection in an individual with a competent immune system generally has minor or no symptoms. The infection tends to be self limiting, with the individual's immune system controlling and eliminating most of the parasites. Some parasites remain in bradyzoite form following acute infection and will be present in cysts in the central nervous system and muscle throughout the remainder of the individual's life.
Toxoplasma gondii is the causative agent in toxoplasmosis, an important disease in immunocompromised patients as well as congenitally-infected human fetuses. In contrast to the mild clinical symptoms of infection seen in a healthy individual with an intact immune system, subjects with weakened or otherwise compromised immune systems can have serious clinical effects from toxoplasma infection. In the fetus, toxoplasma infection can cause mental retardation, visual defects, and death. Toxoplasma infection can cause neurological damage, ocular lesions and death in adults with compromised immune systems, a group that includes for example individuals with HIV infection or patients undergoing immune-suppressive treatment for cancer.
Toxoplasma gondii is also pathogenic to animals, particularly sheep, in which it causes abortion, stillbirth, and fetal mummification. The pathology of toxoplasmosis in its human and animal hosts is a direct result of repeated cycles of host cell invasion, parasite replication, and host cell lysis. In addition, Toxoplasma gondii causes encephalitis, a dangerous life-threatening disease.
Acute toxoplasmosis can be difficult to treat. Sulfadiazine/pyramethamine is a regimen of choice, although side effects serious enough to warrant discontinuation of treatment are common. The toxic and potentially teratogenic effects of this regimen make management of the pregnant woman particularly problematic. AIDS patients require lifelong suppressive therapy to prevent relapse, and as many as one third of the patients receiving suppressive sulfadiazine/pyrimethamine therapy cannot tolerate the adverse side effects. For those who can tolerate the drugs, relapse occurs frequently. Pyrimethamine/clindamycin is a useful alternative therapy in AIDS patients who suffer an unusually high frequency of side effects from sulfa drugs. Unfortunately, this alternative combination can also cause considerable toxicity and is less effective at preventing relapse. Prevention of transmission through vaccination may, in some cases, be preferable to treatment, particularly for pregnant women and the immunocompromised.
The World Health Organization estimates that 300–500 million people are infected by malaria each year and that more than 2 million people, mostly women and children under the age of five, die from malaria annually. The disease has in recent years, made a dramatic comeback in regions where the disease was once eliminated or suppressed. Plasmodium falciparum causes a severe form of human malaria and is responsible for nearly all malaria-specific mortality. Resistance of Plasmodium to anti-malarial drugs is an increasingly serious problem in fighting the disease.
Other Apicomplexan parasitic infections also have severe clinical symptoms and may result in death of humans and livestock. Ticks transmit babesiosis, and although this is primarily a disease of animals, humans are also infected with this parasite. There are over 100 species of Babesia with Babesia microti and Babesia divergens the two most likely to cause human infection. Babesia microti is the organism responsible for a growing number of cases of infection especially in the northeast United States. Babesiosis is not only transmitted via tick bites, it can also be transmitted via blood transfusions, with documented cases of infection via this method.
Sarcocystis parasites may be ingested by humans in undercooked meat, and once in the body, they may form intestinal infections. More commonly, the sporocysts are ingested via fecal contamination, after which the sporocysts may result in cyst formation in striated muscle and cardiac muscle in the host.
Cryptosporidosis is a common infection in subjects with compromised immune systems such as AIDS patients and patients undergoing cancer therapy. Like sarcosporidiosis, the parasites are ingested via fecal contamination of oocysts, which release sporozoites that infect epithelial cells of the intestinal tract resulting in severe and at times life-threatening diarrheal disease. Although symptomatic infection is most likely in immunocompromised individuals, asymptomatic infection also occurs in immunocompetent subjects and the infection is easily passed between individuals.
Theileria infection results in disorders such as: East Coast Fever and Mediterranean Coast Fever, and is transmitted by ticks. Following infection, the parasites are located in the host's red blood cells and clinical symptoms include fever, weight loss, enlarged lymph nodes and spleen, mild anemia, and possible pulmonary involvement.
There are numerous species of Eimeria, and an oral/fecal route of transmission results in intestinal infection in cows, sheep, goats, pigs, ducks, chickens, turkeys, and rabbits, with the domestic chicken host to seven different species of Eimeria. Due to its widespread nature and its effects on the host animal, which may result in sub-optimal weight gain and reduced economic value, Eimeria is an economically important disease in modern poultry production. Eimeria is estimated to have resulted in losses of over 50 million dollars in the United States in 1986 alone.
Another phylum of protozoa, microsporidia, includes obligate, intracellular pathogens, which cause intestinal and systemic infections in immunocompromized patients, as well as economically important infections in fish and invertebrates. Microsporidiosis in patients suffering from acquired immune deficiency syndrome (AIDS) is primarily associated with Encephalitozoon species (including E. intestinalis, E. cuniculi, and E. hellem) and Enterocytozoon bieneusi. Microsporidiosis is a frequent cause of chronic diarrhea in AIDS patients and may also be found outside of the intestine in the eye, biliary tract, nasal sinuses, urinary tract and respiratory tract.
It will be appreciated that there is an urgent need for new chemotherapeutic agents to combat protozoal parasites, which are sufficiently effective, do not have harmful side effects, and are not difficult or expensive to administer. Preferably, the anti-protozoal compounds are active against a broad spectrum of protozoa, while remaining non-toxic to human and other mammalian cells. However, although high-throughput screening assays are playing an increasingly important role in the identification of therapeutic compounds as well as compounds that are useful in biological research, high-throughput screening assays are rare in the field of parisitology, usually due to the complex life cycle of the parasite and the experimental intractability of the system. Current approaches often rely on classical genetic systems, e.g., the identification of temperature sensitive mutants, inducible promoters and the like. Clearly, in order to identify the much needed anti-protozoal agents, there is a need for improved assay systems for identifying these agents. Such assay systems are provided herein below.